Isolated dimeric fibroblast activation protein alpha, and uses thereof

ABSTRACT

The invention involves dimeric forms of the protein known as fibroblast activation protein alpha, or &#34;FAPα&#34; and its uses.

RELATED APPLICATION

This application is a continuation-in-part of Ser. No. 08/230,491, filed Apr. 20, 1994, now U.S. Pat. No. 5,587,299 and incorporated by reference.

FIELD OF THE INVENTION

This invention relates to certain molecules associated with cancer tissues and reactive tumor stromal cells. More particularly, it relates to fibroblast activation protein alpha ("FAPα" hereafter) molecules. A monomeric form of the molecule has previously been identified immunochemically, but nucleic acid molecules coding for it had not been isolated or cloned nor have dimers been identified. These, inter alia, are features of the invention. The monomeric protein has a molecular weight of from about 88 to about 95 kilodaltons as determined by SDS-PAGE of boiled samples. The dimer has a molecular weight of about 170 kilodaltons as determined by SDS-PAGE of unboiled samples. FAPα is characterized by a number of features and properties which are shared by and characteristic of membrane bound enzymes, suggesting very strongly that it, too, is a membrane bound enzyme. The nucleic acid molecules, which are a key part of the invention, are useful both as probes for cells expressing FAPα, and as starting materials for recombinant production of the protein. The FAPα protein can then be used to produce monoclonal antibodies specific for the protein and are thus useful diagnostic agents themselves. They also have additional uses, including uses related to enzymatic functions, as described herein.

BACKGROUND AND PRIOR ART

The invasive growth of epithelial cancers is associated with characteristic cellular and molecular changes in the supporting stroma. For example, epithelial cancers induce the formation of tumor blood vessels, the recruitment of reactive tumor stromal fibroblasts, lymphoid and phagocytic infiltrates, the release of peptide mediators and proteolytic enzymes, and the production of an altered extracellular matrix (ECM). See, e.g., Folkman, Adv. Cancer Res. 43: 175-203 (1985); Basset et al., Nature 348: 699-704 (1990); Denekamp et al., Cancer Metastasis Rev. 9: 267-282 (1990); Cullen et al., Cancer Res. 51: 4978-4985 (1991); Dvorak et al., Cancer Cells 3: 77-85 (1991); Liotta et al., Cancer Res. 51: 5054s-5059s (1991); Garin-Chesa et al., J. Histochem. Cytochem. 37: 1767-1776 (1989). A highly consistent molecular trait of the stroma in several common histologic types of epithelial cancers is induction of the fibroblast activation protein (FAPα), a cell surface glycoprotein with an observed M_(T) of 95,000 originally discovered with a monoclonal antibody, mAb F19, raised against proliferating cultured fibroblasts. See Rettig et al., Cancer Res. 46: 6406-6412 (1986); Rettig et al., Proc. Natl. Acad. Sci. USA 85: 3110-3114 (1988); Garin-Chesa et al., Proc. Natl. Acad. USA 87: 7235-7239 (1990); Rettig et al., Cancer Res. 53: 3327-3335 (1993). Each of these four papers is incorporated by reference in its entirety.

Immunohistochemical studies such as those cited supra have shown that FAPα is transiently expressed in certain normal fetal mesenchymal tissues but that normal adult tissues are generally FAPα⁻. Similarly, malignant epithelial, neural and hematopoietic cells are generally FAPα⁻. However, most of the common types of epithelial cancers, including >90% of breast, lung, skin, pancreas, and colorectal carcinomas, contain abundant FAPα⁺ reactive stromal fibroblasts. Garin-Chesa et al., Proc. Natl. Acad. Sci. USA 87: 7235-7239 (1990). The FAPα⁺ tumor stromal fibroblasts almost invariably accompany tumor blood vessels, forming a distinct cellular compartment interposed between the tumor capillary endothelium and the basal aspect of malignant epithelial cell clusters. While FAPα⁺ stromal fibroblasts are found in both primary and metastatic carcinomas, benign and premalignant epithelial lesions, such as fibroadenomas of the breast and colorectal adenomas only rarely contain FAPα⁺ stromal cells. In contrast to the stroma-specific localization of FAPα in epithelial neoplasms, FAPα is expressed in the malignant cells of a large proportion of bone and soft tissue sarcomas. (Rettig et al., Proc. Natl. Acad. Sci. USA 85: 3110-3114 (1988)). Finally, FAPα⁺ fibroblasts have been detected in the granulation tissue of healing wounds (Garin-Chesa et al., supra). Based on the restricted distribution pattern of FAPα in normal tissues and its uniform expression in the supporting stroma of many epithelial cancers, clinical trials with ¹³¹ I-labeled mAb F19 have been initiated in patients with metastatic colon cancer (Welt et al., Proc. Am. Assoc. Cancer Res. 33: 319 (1992); Welt et al. J. Clin. Oncol. 12: 1561-1571 (1994)) to explore the concept of "tumor stromal targeting" for immunodetection and immunotherapy of epithelial cancers.

Rettig et al., Int. J. Cancer 58: 385-392 (1994), incorporated by reference, discusses the FAPα molecule and its features. Rettig et al postulate that FAPα is found in high molecular weight complexes in excess of 400 kilodaltons, but do not discuss the possibility of dimeric molecules, nor does the paper elaborate on the specific enzymatic properties of the molecule.

The induction of FAPα⁺ fibroblasts at times and sites of tissue remodeling during fetal development, tissue repair, and carcinogenesis is consistent with a fundamental role for this molecule in normal fibroblast physiology. Thus, it is of interest and value to isolate and to clone nucleic acid molecules which code for this molecule. This is one aspect of the invention, which is described in detail together with other features of the invention, in the disclosure which follows. Further aspects of the invention include the dimeric FAPα molecules, and the exploitation of the properties of these molecules. These features are also elaborated upon hereafter.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 compares the deduced amino acid sequence for FAPα, and the known sequence of CD26. The alignment has been optimized SEQ ID NO:2 gives the sequence of FAPα. SEQ ID NO:3 gives the amino acid sequence of CD26.

FIGS. 2A-2H, inclusive, display immunohistochemical detection of FAPα and CD26 in various tissues. In FIGS. 2A and 2B, breast cancer is studied, for FAPα (FIG. 2A), and CD26 (FIG. 2B). In FIGS. 2C and 2D, malignant fibrous histiocytoma is studied, for FAPα (FIG. 2C), and CD26 (FIG. 2D). Dermal scar tissue is examined in FIGS. 2E (FAPα), and 2F (CD26). Renal cell carcinoma is studied in FIGS. 2G (FAPα), and 2H (CD26).

FIG. 3 presents some of the data generated in experiments which showed that FAPα had extracellular matrix (ECM) protein degrading activity. When zymographic detection of gelatin degrading extracts of 293-FAP was carried out, the active substance was found to have a molecular weight of about 170 kD, via SDS-PAGE, using unboiled samples to preserve enzyme activity.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example 1

Fibroblast cell line WI-38 had been observed, previously, to react with mAb F19 (Rettig et al., Canc. Res. 46: 6406-6412 (1986); Rettig et al., Proc. Natl. Acad. USA 85: 3110-3114 (1988); Garin-Chesa et al., Proc. Natl. Acad. Sci. USA 87: 7235-7239 (1990); Rettig et al., Canc. Res. 53: 3327-3335 (1993)). It was used in the experiments which follow.

A cDNA library was prepared from WI-38, using well known techniques and commercially available materials. Specifically, the library was constructed in expression vector pCDNAI, using the Fast Track mRNA isolation kit, and Librarian cDNA phagemid system. Once the library was prepared, the vectors were electroporated into cell line E. coli MC 1061/P3. The pCDNAI expression vector contains an antibiotic resistance gene, so the E. coli were selected via antibiotic resistance. The colonies which were resistant were then used in further experiments. The plasmid DNA from the colonies was obtained via alkaline lysis and purification on CsCl₂, in accordance with Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab, Cold Spring Harbor, N.Y. 2d Ed. 1989). The technique is well known to the art, but is incorporated by reference herein.

Once the plasmid DNA was isolated, it was used to transfect COS-1 cells, which were then cultured for forty-eight hours, after which these were tested with antibody coated dishes. The mAbs used included F19, as described by Rettig et al., (1986), supra, which is incorporated by reference in its entirety. As COS-1 cells are normally FAPα⁻, any positive results indicated the presence of the coding sequence. The immunoselection protocol was that of Aruffo et al., Proc. Natl. Acad. Sci USA 84: 3365-3369 (1987), incorporated by reference herein.

Plasmid DNA from positive clones was recovered, in accordance with Hirt, J. Mol. Biol. 26: 365-369 (1967), reintroduced into E. coli MC 1061/P3, and reselected in COS-1 cells.

The protocol presented herein was followed for four rounds. After this, the plasmid DNA of 50 isolated bacterial colonies was purified, using the Qiagen plasmid kit. Of the colonies, 27 clones were found to contain identical 2.8 kb inserts, as determined by EcoRI restriction enzyme mapping. Several of these were found to contain FAPα-specific cDNA as determined by transient expression in COS-1 cells and direct immunofluorescence staining with mAb F19. One of these clones, i.e., "pFAP.38" was selected for further study, as elaborated upon infra.

Example 2

Once pFAP.38 had been identified, it was tested together with a vector coding for known cell surface marker CD26 ("pCD26"), as well as with control vector pCDNA I.

In these experiments, COS-1 cells were transfected with one of pFAP.38, pCD26, or pCDNAI. After forty-eight hours, the transfectants were tested, using the well known MHA rosetting assay for cell surface antigen expression. In these experiments, mAb F19, which is FAPα specific, was used, together with mAb EF-1, which is CD26 specific. Also used were four other FAPα specific mAbs, i.e., FB23, FB52, FB58 and C48. Also tested were two cancer cell lines, which are known to react with mAb F19 (SW872 liposarcoma), or EF-1 (SK-OV6 ovarian cancer). The results are set forth in Table 1, which follows.

                  TABLE 1                                                          ______________________________________                                         Cell surface expression of multiple FAPα epitopes and                    CD26 in human cells and COS-1 cell transfectants                                           Cell surface antigen expression                                    Target cell   F19    FB23   FB52 FB58 C48  EF-1                                ______________________________________                                         Human cells                                                                    SW872 liposarcoma                                                                            >95%   >95%   >95% >95% >95% --                                  SK-OV6 ovarian cancer                                                                        --     --     --   --   --   >95%                                COS-1 transfectants                                                            COS.pCDNAI control                                                                           --     --     --   --   --   --                                  COS.pFAP 38   40%    30%    40%  20%  20%  --                                  COS.pCD26     --     --     --   --   --   40%                                 ______________________________________                                    

Example 3

Immunoprecipitation studies were then carried out to identify the antigen being targeted by the antibodies.

Cells were metabolically labelled with Trans ³⁵ S-label, (ICN) extracted with lysis buffer (0.01M Tris-HCl/0.15M NaCl/0.01M MgCl₂ /0.5% Nonidet P-40/aprotinin (20 ug/ml)/2 mM phenylmethylsulfonyl fluoride), and then immunoprecipitated. The protocols used are all well known, as will be seen by reference to Rettig et al., Canc. Res. 53: 3327-3335 (1993); and Fellinger et al., Canc. Res. 51: 336-340 (1991), the disclosures of which are all incorporated by reference in their entirety. Precipitating mAbs were negative control mouse Ig, mAb F19, or EF-1. Control tests were carried out with mock transfected COS-1 cells. Following immunoprecipitation, the immunoprecipitates were boiled in extraction buffer and separated by NaDodSO₄ /PAGE, under reducing conditions. In some experiments, an additional test was carried out to determine whether or not the immunoprecipitated material was glycosylated. In these experiments, cell extracts were fractionated with Con A-SEPHAROSE prior to immunoprecipitation. Following immunoprecipitation, but prior to fractionation on NaDodSO₄ /PAGE, these precipitates were digested with N-Glycanase.

The results showed that, in COS-1 cells, pFAP.38 directs expression of an 88 kd protein species (as determined via SDS-PAGE), which is slightly smaller than the 95 kd FAPα species produced by SW872, or cultured fibroblasts. Digestion with N-Glycanase produced peptides of comparable size (i.e., 74 kd versus 75 kd), showing that the glycosylation of the FAPα protein in COS-1 cells is different than in the human cell lines.

Example 4

Classic Northern blot analysis was then carried out, using the mRNA from FAPα⁺ fibroblast cell lines WI-38 and GM 05389, and FAPα⁻ ovarian cancer cell line SK-OV6. Using the procedures of Sambrook et al., supra, five micrograms of mRNA from each cell line were tested. The probes used were ³² P labelled, and were prepared from a 2.3 kb ECO I fragment of pFAP.38, a 2.4 kb Hind III fragment of CD26, and a 1.8 kb BamHI fragment of γ-actin cDNA. These fragments had been purified from 1% agarose gels.

The extracts of FAPα⁺ fibroblast strains showed a 2.8 kb FAP mRNA species, but extracts of SK-OV6 do not. A γ-actin mRNA species (1.8 kb), was observed in all species.

Example 5

The cDNA identified as coding for FAPα was subjected to more detailed analysis, starting with sequencing. The classic Sanger methodology, as set forth in Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977), was used to sequence both strands of the cDNA. Once this was secured, an amino acid sequence was deduced therefrom. This information is presented in SEQ ID NO: 1. The sequence was then compared to the known amino acid sequence of CD26 (Morimoto et al., J. Immunol. 143: 3430-3437 (1989)). FIG. 1 presents the comparison, using optimized sequence alignment. Any gaps in the comparison are indicated by asterisks, while identical amino acids are shown by dashes in the CD26 sequence. A hydrophobic, putative transmembrane sequence is double underlined, while potential N-glycosylation sites are single underlined.

The sequence analysis shows a 2815 base pair insert, wherein 2280 base pairs constitute the open reading frame. This ORF extends from start codon ATG at nucleotide 209, to stop codon TAA at 2486.

The deduced polypeptide is 760 amino acids long, and has a molecular weight of 87,832. In contrast, N-Glycanase digested, immunopurified FAPα was reported to have an estimated M_(T) of 75,000 on NaDodSO₄ /PAGE (Rettig et al., Canc. Res. 53: 3327-3335 (1993)).

A GenBank data base search was carried out. The most closely related genes found were those encoding dipeptidyl peptidase IV homologues (DPPIV; EC 3.4.14.5), with human DPPIV (also known as T-cell activation antigen CD26), showing 51% nucleotide sequence identity, and 52% amino acid sequence identity.

The second set of related genes are human, rat, and bovine homologues of DPPX, a gene of unknown function widely expressed in brain and other normal tissues. The predicted human DPPX gene product shows about 30% amino acid sequence identity with FAPα and CD26. The FAPα molecule exhibits structural features typical of type II integral membrane proteins, including a large COOH-terminal extracellular domain, a hydrophobic transmembrane segment, and a short cytoplasmic tail. The putative extracellular domain contains five potential N-glycosylation sites, eleven cysteine residues (nine of which are conserved between FAPα and CD26), and three segments corresponding to highly conserved catalytic domains characteristic of serine proteases, such as DPPIV. These conserved sequences are presented in Table 2, which follows. Comparisons to DPPIV and DPPX were made via Morimoto et al., supra; Wada et al., Proc. Natl. Acad. Sci. USA 89: 197-201 (1992); Yokotani et al., Human Mol. Genet. 2: 1037-1039 (1993).

                                      TABLE 2                                      __________________________________________________________________________     Putative catalytic domains of FAPα, DPPIV and DPPX.                                 624           702          734                                      Human FAPα                                                                      ....WGWSYGG SEQ ID NO.4........GTADDNV SEQ ID NO.6........DQNHGLS              SEQ ID NO.7....                                                         Human DPPIV                                                                           ....WGWSYGG SEQ ID NO.4........GTADDNV SEQ ID NO.6........DEDHGIA              SEQ ID NO.8....                                                         Mouse DPPIV                                                                           ....WGWSYGG SEQ ID NO.4........GTADDNV SEQ ID NO.6........DEDHGIA              SEQ ID NO.8....                                                         Rat DPPIV                                                                             ....WGWSYGG SEQ ID NO.4........GTADDNV SEQ ID NO.6........DEDHGIA              SEQ ID NO.8....                                                         Yeast DPPIV                                                                           ....FGWSYGG SEQ ID NO.4........GTGDDNV SEQ ID NO.6........DSDHSIR              SEQ ID NO.8....                                                         Human DPPX                                                                            ....FGKDYGG SEQ ID NO.5........PTADEKI SEQ ID NO.9........DESHYFT              SEQ ID NO.10....                                                        Rat DPPX                                                                              ....FGKDYGG SEQ ID NO.6........ATADEKI SEQ ID NO.9........DESHYFH              SEQ ID NO.10....                                                        Bovine DPPX                                                                           ....FGKDYGG SEQ ID NO.5........ATADEKI SEQ ID NO.9........DESHYFS              SEQ ID NO.10....                                                        __________________________________________________________________________

Example 6

An additional set of experiments were carried out to determine whether FAPα related sequences are present in non-human species. To do so, human, mouse, and Chinese hamster genomic DNA was digested using restriction enzymes, and tested, via Southern blotting, using the 2.3 kb fragment, labelled with ³² P, described supra. Hybridization was carried out using stringent washing conditions (0.1×SSC, 0.1% NaDodSO₄, 68° C.). Cross-hybridization was readily observed with both the mouse and hamster DNA, suggesting the existence of highly conserved FAPα homologues. In control experiments using the CD26 cDNA fragment described supra, no evidence of cross hybridization was observed.

Example 7

The CD26 molecule shares a number of biochemical and serological properties with FAPβ, which is a previously described, FAPα associated molecule having a molecular weight of 105 kD, and is found on cultured fibroblasts and melanocytes (Rettig et al., Canc. Res. 53: 3327-3335 (1993)). Cotransfection experiments were carried out to determine whether FAPβ is a CD26 gene product. To test this, the same protocols were used which were used for transfection with pFAP.38 or pCD26, as described supra, but using the two vectors. The results presented supra showed that cotransfection efficiency was about 40% for each vector, so about 10-20% of cell should be cotransfected.

Following cotransfection, the COS-1 cells were Trans ³⁵ S-labeled, as described supra, then lysed, also as described supra.

The resulting cell extracts were separated on Con A SEPHAROSE, and the antigen (FAPα and/or CD26) were recovered in the Con A-bound fraction. The bound fraction was eluted with 0.25M α-D-mannopyranoside. Immunoprecipitation was then carried out, as described supra, and the precipitates were separated on NaDodSO₄ /PAGE, also as discussed supra.

Those cells transfected only with pFAP.38 produced FAPα, but not FAPβ (determined from mAb F19 immunoprecipitates). They also produce no CD26 antigen (tested with EF-1). Those cells transfected with pCD26 alone produce CD26 but no FAPα. Cotransfectants produce CD26 and FAPα/FAPβ heteromers, as determined in the mAb F19 precipitates. This result provides direct evidence that FAPβ is a CD26 gene product.

Example 8

It has been observed previously that some cultured human cell types coexpress FAPα and CD26, and show FAPα/CD26 heteromer formation. In vivo distribution patterns of FAPα and CD26, however, as determined in previous immunohistochemical studies, appeared to be non-overlapping. (See Rettig et al., Proc. Natl. Acad. Sci. USA 85: 3110-3114 (1988); Garin-Chesa et al., Proc. Natl. Acad. Sci. USA 87: 7235-7329 (1990); Rettig et al., Canc. Res. 53: 3327-3335 (1993); Stein et al., in Knapp et al., eds. Leukocyte typing IV-white cell differentiation antigens, pp. 412-415 (Oxford University Press, N.Y. 1989), pp. 412-415; Mobious et al., J. Exp. Immunol. 74: 431-437 (1988)). In view of the potential significance of FAPα/CD26 coassociation, tissue distribution was reexamined, via side by side immunohistochemical staining of normal tissues and lesional tissues known to contain FAPα⁺ fibroblasts or FAPα⁺ malignant cells.

To test the samples, they were embedded in OCT compound, frozen in isopentane precooled in liquid nitrogen, and stored at -70° C. until used. Five micrometer thick sections were cut, mounted on poly-L-lysine coated slides, air dried, and fixed in cold acetone (4° C., for 10 minutes). The sections were then tested with mAbs (10-20 ug/ml), using the well known avidin-biotin immmunoperoxidase method, as described by, e.g., Garin-Chesa et al., J. Histochem. Cytochem. 37: 1767-1776 (1989); Garin-Chesa et al., Proc. Natl. Acad. Sci. USA 87: 7235-7239 (1990); Rettig et al., Canc. Res. 53: 3327-3335 (1993); Garin-Chesa et al., Am. J. Pathol. 142: 557-567.

The results are shown in FIG. 2. Breast, colorectal, pancreas and lung carcinomas showed strong expression of FAPα and no CD26 was found (see FIGS. 2A and 2B). Five FAPα⁺ sarcomas, including malignant fibrous histiocytoma (FIGS. 2C and 2D), were tested, and there was no expression of CD26. Examination of reactive fibroblasts of healing dermal wounds (FIGS. 2E, 2F), showed abundant expression of both FAPα and CD26. The three renal carcinomas tested (FIGS. 2G, 2H), showed expression of CD26 in malignant epithelium. FAPα was absent from malignant epithelial cells, and showed low expression in the stroma of these carcinomas.

Example 9

A mammalian cell line, transfected with a FAPα encoding cDNA, was prepared.

Human embryonic kidney cell line 293 is well known and widely available from, e.g., the American Type Culture Collection.

Samples of 293 were maintained, in an incubator, at 37° C., in an atmosphere of 95% air, and 5% CO₂. The cells were cultured in a 50:50 mixture of Dulbecco's modified minimal essential medium and Ham's F12 medium, augmented with 10% fetal bovine serum, penicillin and streptomycin. Following the procedures described by Ustar et al., Eur. Mol. Biol. J. 1991, and/or Park et al., J. Biol. Chem. 169: 25646-25654 (1994), both of which are incorporated by reference, cDNA for FAPα (i.e., SEQ ID NO: 1), was transfected into the 293 cells. Details of the cDNA vector are provided, supra (pFAP.38). Transfectants were selected for resistance to antibiotics (200 ug/ml Geneticin), and were then maintained in selection medium, containing Geneticin.

Individual colonies of resistant cells were picked, grown to confluence in 6 well tissue culture plates, and were tested for FAPα expression in an immunofluorescence assay (IFA), using FAPαspecific monoclonal antibody F19 as described supra.

Those colonies which expressed FAPα were expanded, and monitored by indirect IFA and cytofluorometric analysis, also as set forth, supra.

The IFAs were positive for the transfectants, referred to hereafter as cell line 293-FAP, but were negative for parental line 293.

Example 10

In order to confirm that recombinant FAPα was, in fact, being produced, a series of immunoprecipitation experiments were carried out. These followed the methods of Park, et al., supra, and Rettig et al., Canc. Res. 53: 3327-3335 (1993), both of which are incorporated by reference. Essentially, ³⁵ S! methionine labelled cell extracts were combined with monoclonal antibody F19, in the manner described supra. Precipitates were then boiled in extraction buffer and run on SDS-PAGE gels, using, as a negative control, mouse IgG1. Both cell line 293-FAP, and non transfected line 293 were tested. The results indicated clearly, that recombinant FAPα was produced by the transfected cell line 293-FAP. This was determined by immunoprecipitation analyses, using FAPα specific monoclonal antibody F19.

Example 11

The ability to produce recombinant FAPα permitted further study of the molecule's properties. Specifically, given the structural features outlined in the prior examples, experiments were designed to determine if FAPα possesses enzymatic activities. The experiments were designed to test whether or not FAPα had extracellular matrix (ECM) protein degrading activity.

Extracts of 293-FAP cells were prepared, using an extraction buffer (0.15M NaCl, 0.05M Tris-HCl, pH 7.4, 10 mM MgCl₂, 1 percent Triton X-114), were cleared by centrifugation (4,000×g, 10 minutes at 4° C.), and phase partitioned at 37° C. for 10-20 minutes. This was followed by further centrifugation (4000×g, 20 minutes at 20°-25° C.). Detergent phases were diluted with buffer (0.15M NaCl, 0.05M Tris-HCl pH 7.4, 5 mM CaCl₂, 5 mM MgCl₂, 0.75% Empigen BB), and separated on concanavalin A-Sepharose following Rettig et al., supra. Any concanavalin A bound fractions were eluted with 0.25M methyl-α-D-mannopyranoside in elution buffer 0.15M NaCl, 0.05M Tris-HCl, pH 7.4, 5 mM CaCl₂, 5 mM MgCl₂, 0.1% Triton X-100), mixed with zymography sample buffer (0.25M Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 0.01% bromophenol blue), at a 3:1 ratio, and used for further analysis.

Aliquots of sample were loaded onto polyacrylamide gels containing 0.1% of either of gelatin or casein. Electrophoresis was then carried out in a Biorad Mini-Protein II system, at 20 mA constant current for 1.5-2 hours, until the bromophenol blue dye fronts of samples had reached the lower end of the gel. The gel was removed and incubated for one hour at 20°-25° C. in a 2.5% aqueous solution of Triton X-100 on a rotary shaker. The Triton X-100 solution was decanted, and replaced with enzyme buffer (0.05M Tris-HCl, pH 7.5, 0.2M NaCl, 5 mM CaCl₂, 5 mM MgCl₂, 0.02% Brij 35). The gel was then incubated at 37° C. or 41° C., followed by staining or destaining at room temperature. Gels were stained with 0.5% of Coomassie Brilliant Blue G-250 in an aqueous solution of 30% methanol and 10% acetic acid for 15, 30, and 60 minutes, respectively. Subsequently, gels were incubated for 15 minutes in an aqueous solution of 30% CH₃ OH and 5% glycerol, followed by drying between sheets of cellophane.

Gelatinase activity was evaluated in accordance with Kleiner et al., Anal. Biochem. 218: 325-329 (1994), incorporated by reference in its entirety. This is a routine assay used to determine whether or not a protease capable of digesting gelatin is present. Labelled molecular weight standard were run on the same gels, under reducing conditions, for molecular weight determinations.

Proteolytic activity for defined amino acid sequence motifs were tested, using a well known membrane overlay assay. See Smith et al, Histochem. J. 24(9): 637-647 (1992), incorporated by reference. Substrates were Ala-Pro-7-amino-4-trifluoromethyl coumarin, Gly-Pro-7-amino-4-trifluoromethyl coumarin, and Lys-Pro-7-amino-4-trifluromethyl coumarin.

The results of these experiments are depicted, in part, in FIG. 3. This figure shows zymographic detection of gelatin degrading activity, in the cell extracts. See Kleiner et al., supra. A protein species of approximately 170 kilodaltons, as determined by SDS-PAGE, was observed to have gelatin degrading activity. This species, which was found in the 293-FAP cell line, but not in untransfected 293 cells, is thus identified as FAPα. The molecular weight is consistent with a dimer, i.e., a dimeric FAPα molecule.

The proteolytic activity described herein where gelatin is the substrate was not observed when casein was the substrate.

Example 12

Further studies were then undertaken in order to characterize the 170 kD FAPα dimer further. Specifically, the experiments described in example 11 were repeated, except that 5% of 2-mercaptoethanol or 5 um iodoacetamide was added to the extracts prior to SDS-PAGE, or ethylenediamine N,N,N',N'-tetraacetic acid (10 mM) was added to the incubation buffer used for gelatin zymography. None of these treatments abolished the enzymatic activity. In contrast, heating at 100° C. for five minutes prior to SDS-polyacrylamide gel electrophoresis abolished the gelatin-degrading activity.

Further work, using a membrane overlay assay, described by, e.g., Smith et al., Histochem J. 24(9): 643-647 (1992), incorporated by reference, revealed that the FAPα dimers were able to cleave all of the Ala-Pro, Gly-Pro, and Lys-Pro dipeptides tested.

In further experiments, a fusion protein was produced which comprised the extracellular domains of both FAPα and murine CD8 proteins. This chimeric protein was produced in a baculovirus system in insect cells. The chimeric protein exhibited the same enzymatic activity as FAPα, using the model discussed supra.

The foregoing examples describe an isolated nucleic acid molecule which codes for fibroblast activating protein alpha ("FAPα"), as well as dimeric forms of the molecule, and uses thereof. The expression product of the sequence in COS-1 is a protein which, on SDS-PAGE of boiled samples, shows a molecular weight of about 88 kd. Deduced amino acid sequence, as provided in SEQ ID NO: 1, for one form of the molecule, yields a molecular weight of about 88 kd.

It should be noted that there is an apparent discrepancy in molecular weight in that the COS-1 isolate is glycosylated, while molecular weight from deduced amino acid sequences does not account for glycosylation. Membrane proteins are known to exhibit aberrant migration in gel systems, however, which may explain the difference observed here.

Also a part of the invention are chimeric and fusion proteins, which comprise a portion of FAPα which contain the molecule's catalytic domain, and additional, non FAPα components. The FAPα catalytic domain per se is also a part of the invention.

It is to be understood that, as described, FAPα may be glycosylated, with the type and amount of glycosylation varying, depending upon the type of cell expressing the molecule. The experiment described herein shows this. This is also true for the dimeric form of the molecule, first described herein, having a molecular weight of about 170 kilodaltons as determined by SDS-PAGE of unboiled samples.

The invention also comprehends the production of expression vectors useful in producing the FAPα molecule. In their broadest aspect, these vectors comprise the entire FAPα coding sequence or portions thereof, operably linked to a promoter. Additional elements may be a part of the expression vector, such as protein domains fused to the FAPα protein or protein portions ("fusion protein") genes which confer antibiotic resistance, amplifiable genes, and so forth.

The coding sequences and vectors may also be used to prepare cell lines, wherein the coding sequence or expression vector is used to transfect or to transform a recipient host. The type of cell used may be prokaryotic, such as E. coli, or eukaryotes, such as yeast, CHO, COS, or other cell types.

The identification of nucleic acid molecules such as that set forth in SEQ ID NO: 1 also enables the artisan to identify and to isolate those nucleic acid molecules which hybridize to it under stringent conditions. "Stringent condition" as used herein, refers to those parameters set forth supra, whereby both murine and hamster sequences were also identified. It will be recognized by the skilled artisan that these conditions afford a degree of stringency which can be achieved using parameters which vary from those recited. Such variance is apprehended by the expression "stringent conditions".

The ability of nucleic acid molecules to hybridize to complementary molecules also enables the artisan to identify cells which express FAPα, via the use of a nucleic acid hybridization assay. One may use the sequences described in the invention to hybridize to complementary sequences, and thus identify them. In this way, one can target mRNA, e.g., which is present in any cell expressing the FAPα molecule.

It is of course understood that the nucleic acid molecules of the invention are also useful in the production of recombinant FAPα, in both monomeric and dimeric form. The examples clearly show that host cells are capable of assembling the dimeric forms. The recombinant protein may be used, e.g., as a source of an immunogen for generation of antibodies akin to known mAb F19, and with the same uses. Similarly, the recombinant protein, and/or cells which express the molecule on their surface, may be used in assays to determine antagonists, agonists, or other molecules which interact with the FAPα molecule. Such molecules may be, but are not necessarily limited to, substrates, inhibiting molecules, antibodies, and so forth. This last feature of the invention should be considered in light of the observed structural resemblances to membrane bound enzymes. This type of molecule is associated with certain properties which need not be described in detail here. It will suffice to say that inhibition or potentiation of these properties as associated with FAPα is a feature of this invention. For example, one may identify substrates or the substrate for FAPα molecules, via the use of recombinant cells or recombinant FAPα per se. The substrates can be modified to improve their effect, to lessen their effect, or simply to label them with detectable signals so that they can be used, e.g., to identify cells which express FAPα. Study of the interaction of substrate and FAPα, as well as that between FAPα and any molecule whatsoever, can be used to develop and/or to identify agonists and antagonists of the FAPα molecule.

Also a feature of the invention are isolated, dimeric FAPα molecules which have a molecular weight of about 170 kilodaltons as determined by SDS-PAGE, their use as an enzymatic cleaving agent, and other uses as are described herein. Enzymatically active forms of FAPα may also be produced as recombinant fusion proteins, comprising the catalytic domain of FAPα and other protein domains with suitable biochemical properties, including secretory signals protease cleavage sites, tags for purification, and other elements known to the artisan. The fact that FAPα has particular properties, as described herein, permits the identification of the molecule on cells expressing them. In turn, because the FAPα molecule is associated with tumors and tumor stromal cells, targeting of FAPα with therapeutic agents serves as a way to treat cancerous or precancerous condition, by administering sufficient therapeutic agent to alleviate cancer load.

The experiments showing the proteolytic properties of FAPα lead to yet a further aspect of the invention. It is well known that proteases which degrade extracellular matrix, or "ECM" proteins have an important role on certain aspects of tumor growth, including their effect on tumor cell invasion, tumor blood vessel formation (i.e., neoangiogenesis), and tumor metastasis. Collagens are of special interest vis-a-vis the substrates of proteases, as the collagens are an important part of the ECM. The fact that FAPα digests ECM suggests a therapeutic role for inhibitors of the molecule. "Inhibitors", as used herein, refers to molecules which interfere with FAPα enzyme function. Specifically excluded from such inhibitors is the monoclonal antibody F19. This mAb is known to bind to but not inhibit the enzyme function of FAPα, and hence it is not an inhibitor. The art is quite well versed with respect to monoclonal antibodies which both bind to and inhibit enzymes. Further examples of such inhibitors would include, e.g., substrate derivatives, such as modified collagen molecules, which interfere with the active site or sites of the FAPα molecule. Other suitable inhibitors will be apparent to the skilled artisan, and need not be listed here. In addition, the recombinant FAPα proteins and FAPα-transfected cell lines described supra can be employed in an enzymatic screening assay, using the substrate described supra or other suitable substrates, to identify inhibitors from any compound library,

Other aspects of the invention will be clear to the skilled artisan, and need not be set forth here.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, it being recognized that various modifications are possible within the scope of the invention.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 10                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2815 Base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: double                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                       AAGAACGCCCCCAAAATCTGTTTCTAATTTTACAGAAATCTTTTGAAACTTGGCACGGTA60                 TTCAAAAGTCCGTGGAAAGAAAAAAACCTTGTCCTGGCTTCAGCTTCCAACTACAAAGAC120                AGACTTGGTCCTTTTCAACGGTTTTCACAGATCCAGTGACCCACGCTCTGAAGACAGAAT180                TAGCTAACTTTCAAAAACATCTGGAAAAATGAAGACTTGGGTAAAAATCGTATTTGGAGT240                TGCCACCTCTGCTGTGCTTGCCTTATTGGTGATGTGCATTGTCTTACGCCCTTCAAGAGT300                TCATAACTCTGAAGAAAATACAATGAGAGCACTCACACTGAAGGATATTTTAAATGGAAC360                ATTTTCTTATAAAACATTTTTTCCAAACTGGATTTCAGGACAAGAATATCTTCATCAATC420                TGCAGATAACAATATAGTACTTTATAATATTGAAACAGGACAATCATATACCATTTTGAG480                TAATAGAACCATGAAAAGTGTGAATGCTTCAAATTACGGCTTATCACCTGATCGGCAATT540                TGTATATCTAGAAAGTGATTATTCAAAGCTTTGGAGATACTCTTACACAGCAACATATTA600                CATCTATGACCTTAGCAATGGAGAATTTGTAAGAGGAAATGAGCTTCCTCGTCCAATTCA660                GTATTTATGCTGGTCGCCTGTTGGGAGTAAATTAGCATATGTCTATCAAAACAATATCTA720                TTTGAAACAAAGACCAGGAGATCCACCTTTTCAAATAACATTTAATGGAAGAGAAAATAA780                AATATTTAATGGAATCCCAGACTGGGTTTATGAAGAGGAAATGCTTCCTACAAAATATGC840                TCTCTGGTGGTCTCCTAATGGAAAATTTTTGGCATATGCGGAATTTAATGATAAGGATAT900                ACCAGTTATTGCCTATTCCTATTATGGCGATGAACAATATCCTAGAACAATAAATATTCC960                ATACCCAAAGGCTGGAGCTAAGAATCCCGTTGTTCGGATATTTATTATCGATACCACTTA1020               CCCTGCGTATGTAGGTCCCCAGGAAGTGCCTGTTCCAGCAATGATAGCCTCAAGTGATTA1080               TTATTTCAGTTGGCTCACGTGGGTTACTGATGAACGAGTATGTTTGCAGTGGCTAAAAAG1140               AGTCCAGAATGTTTCGGTCCTGTCTATATGTGACTTCAGGGAAGACTGGCAGACATGGGA1200               TTGTCCAAAGACCCAGGAGCATATAGAAGAAAGCAGAACTGGATGGGCTGGTGGATTCTT1260               TGTTTCAAGACCAGTTTTCAGCTATGATGCCATTTCGTACTACAAAATATTTAGTGACAA1320               GGATGGCTACAAACATATTCACTATATCAAAGACACTGTGGAAAATGCTATTCAAATTAC1380               AAGTGGCAAGTGGGAGGCCATAAATATATTCAGAGTAACACAGGATTCACTGTTTTATTC1440               TAGCAATGAATTTGAAGAATACCCTGGAAGAAGAAACATCTACAGAATTAGCATTGGAAG1500               CTATCCTCCAAGCAAGAAGTGTGTTACTTGCCATCTAAGGAAAGAAAGGTGCCAATATTA1560               CACAGCAAGTTTCAGCGACTACGCCAAGTACTATGCACTTGTCTGCTACGGCCCAGGCAT1620               CCCCATTTCCACCCTTCATGATGGACGCACTGATCAAGAAATTAAAATCCTGGAAGAAAA1680               CAAGGAATTGGAAAATGCTTTGAAAAATATCCAGCTGCCTAAAGAGGAAATTAAGAAACT1740               TGAAGTAGATGAAATTACTTTATGGTACAAGATGATTCTTCCTCCTCAATTTGACAGATC1800               AAAGAAGTATCCCTTGCTAATTCAAGTGTATGGTGGTCCCTGCAGTCAGAGTGTAAGGTC1860               TGTATTTGCTGTTAATTGGATATCTTATCTTGCAAGTAAGGAAGGGATGGTCATTGCCTT1920               GGTGGATGGTCGAGGAACAGCTTTCCAAGGTGACAAACTCCTCTATGCAGTGTATCGAAA1980               GCTGGGTGTTTATGAAGTTGAAGACCAGATTACAGCTGTCAGAAAATTCATAGAAATGGG2040               TTTCATTGATGAAAAAAGAATAGCCATATGGGGCTGGTCCTATGGAGGATACGTTTCATC2100               ACTGGCCCTTGCATCTGGAACTGGTCTTTTCAAATGTGGTATAGCAGTGGCTCCAGTCTC2160               CAGCTGGGAATATTACGCGTCTGTCTACACAGAGAGATTCATGGGTCTCCCAACAAAGGA2220               TGATAATCTTGAGCACTATAAGAATTCAACTGTGATGGCAAGAGCAGAATATTTCAGAAA2280               TGTAGACTATCTTCTCATCCACGGAACAGCAGATGATAATGTGCACTTTCAAAACTCAGC2340               ACAGATTGCTAAAGCTCTGGTTAATGCACAAGTGGATTTCCAGGCAATGTGGTACTCTGA2400               CCAGAACCACGGCTTATCCGGCCTGTCCACGAACCACTTATACACCCACATGACCCACTT2460               CCTAAAGCAGTGTTTCTCTTTGTCAGACTAAAAACGATGCAGATGCAAGCCTGTATCAGA2520               ATCTGAAAACCTTATATAAACCCCTCAGACAGTTTGCTTATTTTATTTTTTATGTTGTAA2580               AATGCTAGTATAAACAAACAAATTAATGTTGTTCTAAAGGCTGTTAAAAAAAAGATGAGG2640               ACTCAGAAGTTCAAGCTAAATATTGTTTACATTTTCTGGTACTCTGTGAAAGAAGAGAAA2700               AGGGAGTCATGCATTTTGCTTTGGACACAGTGTTTTATCACCTGTTCATTTGAAGAAAAA2760               TAATAAAGTCAGAAGTTCAAAAAAAAAAAAAAAAAAAAAAAAAGCGGCCGCTCGA2815                    (2) INFORMATION FOR SEQ ID NO: 2:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 760 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                       MetLysThrTrpValLysIleValPheGlyValAlaThrSerAlaVal                               51015                                                                          LeuAlaLeuLeuValMetCysIleValLeuArgProSerArgValHis                               202530                                                                         AsnSerGluGluAsnThrMetArgAlaLeuThrLeuLysAspIleLeu                               354045                                                                         AsnGlyThrPheSerTyrLysThrPhePheProAsnTrpIleSerGly                               505560                                                                         GlnGluTyrLeuHisGlnSerAlaAspAsnAsnIleValLeuTyrAsn                               65707580                                                                       IleGluThrGlyGlnSerTyrThrIleLeuSerAsnArgThrMetLys                               859095                                                                         SerValAsnAlaSerAsnTyrGlyLeuSerProAspArgGlnPheVal                               100105110                                                                      TyrLeuGluSerAspTyrSerLysLeuTrpArgTyrSerTyrThrAla                               115120125                                                                      ThrTyrTyrIleTyrAspLeuSerAsnGlyGluPheValArgGlyAsn                               130135140                                                                      GluLeuProArgProIleGlnTyrLeuCysTrpSerProValGlySer                               145150155160                                                                   LysLeuAlaTyrValTyrGlnAsnAsnIleTyrLeuLysGlnArgPro                               165170175                                                                      GlyAspProProPheGlnIleThrPheAsnGlyArgGluAsnLysIle                               180185190                                                                      PheAsnGlyIleProAspTrpValTyrGluGluGluMetLeuProThr                               195200205                                                                      LysTyrAlaLeuTrpTrpSerProAsnGlyLysPheLeuAlaTyrAla                               210215220                                                                      GluPheAsnAspLysAspIleProValIleAlaTyrSerTyrTyrGly                               225230235240                                                                   AspGluGlnTyrProArgThrIleAsnIleProTyrProLysAlaGly                               245250255                                                                      AlaLysAsnProValValArgIlePheIleIleAspThrThrTyrPro                               260265270                                                                      AlaTyrValGlyProGlnGluValProValProAlaMetIleAlaSer                               275280285                                                                      SerAspTyrTyrPheSerTrpLeuThrTrpValThrAspGluArgVal                               290295300                                                                      CysLeuGlnTrpLeuLysArgValGlnAsnValSerValLeuSerIle                               305310315320                                                                   CysAspPheArgGluAspTrpGlnThrTrpAspCysProLysThrGln                               325330335                                                                      GluHisIleGluGluSerArgThrGlyTrpAlaGlyGlyPhePheVal                               340345350                                                                      SerArgProValPheSerTyrAspAlaIleSerTyrTyrLysIlePhe                               355360365                                                                      SerAspLysAspGlyTyrLysHisIleHisTyrIleLysAspThrVal                               370375380                                                                      GluAsnAlaIleGlnIleThrSerGlyLysTrpGluAlaIleAsnIle                               385390395400                                                                   PheArgValThrGlnAspSerLeuPheTyrSerSerAsnGluPheGlu                               405410415                                                                      GluTyrProGlyArgArgAsnIleTyrArgIleSerIleGlySerTyr                               420425430                                                                      ProProSerLysLysCysValThrCysHisLeuArgLysGluArgCys                               435440445                                                                      GlnTyrTyrThrAlaSerPheSerAspTyrAlaLysTyrTyrAlaLeu                               450455460                                                                      ValCysTyrGlyProGlyIleProIleSerThrLeuHisAspGlyArg                               465470475480                                                                   ThrAspGlnGluIleLysIleLeuGluGluAsnLysGluLeuGluAsn                               485490495                                                                      AlaLeuLysAsnIleGlnLeuProLysGluGluIleLysLysLeuGlu                               500505510                                                                      ValAspGluIleThrLeuTrpTyrLysMetIleLeuProProGlnPhe                               515520525                                                                      AspArgSerLysLysTyrProLeuLeuIleGlnValTyrGlyGlyPro                               530535540                                                                      CysSerGlnSerValArgSerValPheAlaValAsnTrpIleSerTyr                               545550555560                                                                   LeuAlaSerLysGluGlyMetValIleAlaLeuValAspGlyArgGly                               565570575                                                                      ThrAlaPheGlnGlyAspLysLeuLeuTyrAlaValTyrArgLysLeu                               580585590                                                                      GlyValTyrGluValGluAspGlnIleThrAlaValArgLysPheIle                               595600605                                                                      GluMetGlyPheIleAspGluLysArgIleAlaIleTrpGlyTrpSer                               610615620                                                                      TyrGlyGlyTyrValSerSerLeuAlaLeuAlaSerGlyThrGlyLeu                               625630635640                                                                   PheLysCysGlyIleAlaValAlaProValSerSerTrpGluTyrTyr                               645650655                                                                      AlaSerValTyrThrGluArgPheMetGlyLeuProThrLysAspAsp                               660665670                                                                      AsnLeuGluHisTyrLysAsnSerThrValMetAlaArgAlaGluTyr                               675680685                                                                      PheArgAsnValAspTyrLeuLeuIleHisGlyThrAlaAspAspAsn                               690695700                                                                      ValHisPheGlnAsnSerAlaGlnIleAlaLysAlaLeuValAsnAla                               705710715720                                                                   GlnValAspPheGlnAlaMetTrpTyrSerAspGlnAsnHisGlyLeu                               725730735                                                                      SerGlyLeuSerThrAsnHisLeuTyrThrHisMetThrHisPheLeu                               740745750                                                                      LysGlnCysPheSerLeuSerAsp                                                       755760                                                                         (2) INFORMATION FOR SEQ ID NO: 3:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 766 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                       MetLysThrProTrpLysValLeuLeuGlyLeuLeuGlyAlaAlaAla                               51015                                                                          LeuValThrIleIleThrValProValValLeuLeuAsnLysGlyThr                               202530                                                                         AspAspAlaThrAlaAspSerArgLysThrTyrThrLeuThrAspTyr                               354045                                                                         LeuLysAsnThrTyrArgLeuLysLeuTyrSerLeuArgTrpIleSer                               505560                                                                         AspHisGluTyrLeuTyrLysGlnGluAsnAsnIleLeuValPheAsn                               65707580                                                                       AlaGluTyrGlyAsnSerSerValPheLeuGluAsnSerThrPheAsp                               859095                                                                         GluPheGlyHisSerIleAsnAspTyrSerIleSerProAspGlyGln                               100105110                                                                      PheIleLeuLeuGluTyrAsnTyrValLysGlnTrpArgHisSerTyr                               115120125                                                                      ThrAlaSerTyrAspIleTyrAspLeuAsnLysArgGlnLeuIleThr                               130135140                                                                      GluGluArgIleProAsnAsnThrGlnTrpValThrTrpSerProVal                               145150155165                                                                   GlyHisLysLeuAlaTyrValTrpAsnAsnAspIleTyrValLysIle                               170175180                                                                      GluProAsnLeuProSerTyrArgIleThrTrpThrGlyLysGluAsp                               185190195                                                                      IleIleTyrAsnGlyIleThrAspTrpValTyrGluGluGluValPhe                               200205210                                                                      SerAlaTyrSerAlaLeuTrpTrpSerProAsnGlyThrPheLeuAla                               215220225                                                                      TyrAlaGlnPheAsnAspThrGluValProLeuIleGluTyrSerPhe                               230235240245                                                                   TyrSerAspGluSerLeuGlnTyrProLysThrValArgValProTyr                               250255260                                                                      ProLysAlaGlyAlaValAsnProThrValLysPhePheValValAsn                               265270275                                                                      ThrAspSerLeuSerSerValThrAsnAlaThrSerIleGlnIleThr                               280285290                                                                      AlaProAlaSerMetLeuIleGlyAspHisTyrLeuCysAspValThr                               295300305                                                                      TrpAlaThrGlnGluArgIleSerLeuGlnTrpLeuArgArgIleGln                               310315320325                                                                   AsnTyrSerValMetAspIleCysAspTyrAspGluSerSerGlyArg                               330335340                                                                      TrpAsnCysLeuValAlaArgGlnHisIleGluMetSerThrThrGly                               345350355                                                                      TrpValGlyArgPheArgProSerGluProHisPheThrLeuAspGly                               360365370                                                                      AsnSerPheTyrLysIleIleSerAsnGluGluGlyTyrArgHisIle                               375380385                                                                      CysTyrPheGlnIleAspLysLysAspCysThrPheIleThrLysGly                               390395400405                                                                   ThrTrpGluValIleGlyIleGluAlaLeuThrSerAspTyrLeuTyr                               410415420                                                                      TyrIleSerAsnGluTyrLysGlyMetProGlyGlyArgAsnLeuTyr                               425430435                                                                      LysIleGlnLeuSerAspTyrThrLysValThrCysLeuSerCysGlu                               440445450                                                                      LeuAsnProGluArgCysGlnTyrTyrSerValSerPheSerLysGlu                               455460460                                                                      AlaLysTyrTyrGlnLeuArgCysSerGlyProGlyLeuProLeuTyr                               465470475480                                                                   ThrLeuHisSerSerValAsnAspLysGlyLeuArgValLeuGluAsp                               485490495                                                                      AsnSerAlaLeuAspLysMetLeuGlnAsnValGlnMetProSerLys                               500505510                                                                      LysLeuAspPheIleIleLeuAsnGluThrLysPheTrpTyrGlnMet                               515520525                                                                      IleLeuProProHisPheAspLysSerLysLysTyrProLeuLeuLeu                               530535540                                                                      AspValTyrAlaGlyProCysSerGlnLysAlaAspThrValPheArg                               545550555560                                                                   LeuAsnTrpAlaThrTyrLeuAlaSerThrGluAsnIleIleValAla                               565570575                                                                      SerPheAspGlyArgGlySerGlyTyrGlnGlyAspLysIleMetHis                               580585590                                                                      AlaIleAsnArgArgLeuGlyThrPheGluValGluAspGlnIleGlu                               595600605                                                                      AlaAlaArgGlnPheSerLysMetGlyPheValAspAsnLysArgIle                               610615620                                                                      AlaIleTrpGlyTrpSerTyrGlyGlyTyrValThrSerMetValLeu                               625630635640                                                                   GlySerGlySerGlyValPheLysCysGlyIleAlaValAlaProVal                               645650655                                                                      SerArgTrpGluTyrTyrAspSerValTyrThrGluArgTyrMetGly                               660665670                                                                      LeuProThrProGluAspAsnLeuAspHisTyrArgAsnSerThrVal                               675680685                                                                      MetSerArgAlaGluAsnPheLysGlnValGluTyrLeuLeuIleHis                               690695700                                                                      GlyThrAlaAspAspAsnValHisPheGlnGlnSerAlaGlnIleSer                               705710715720                                                                   LysAlaLeuValAspValGlyValAspPheGlnAlaMetTrpTyrThr                               725730735                                                                      AspGluAspHisGlyIleAlaSerSerThrAlaHisGlnHisIleTyr                               740745750                                                                      ThrHisMetSerHisPheIleLysGlnCysPheSerLeuPro                                     755760765                                                                      (2) INFORMATION FOR SEQ ID NO: 4:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (D) OTHER INFORMATION: The first Xaa is either Trp or Phe.                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                       XaaGlyTrpSerTyrGlyGly                                                          (2) INFORMATION FOR SEQ ID NO: 5:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                       PheGlyLysAspTyrGlyGly                                                          5                                                                              (2) INFORMATION FOR SEQ ID NO: 6:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (D) OTHER INFORMATION: Xaa is either Ala or Gly                                (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                       GlyThrXaaAspAspAsnVal                                                          5                                                                              (2) INFORMATION FOR SEQ ID NO: 7:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                       AlaGlnAsnHisGlyLeuSer                                                          5                                                                              (2) INFORMATION FOR SEQ ID NO: 8:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (D) OTHER INFORMATION:                                                         The first Xaa is Glu or Ser. When the first Xaa is Glu,                        the second Xaa is Gly and the third is Ala. When the                           first Xaa is Ser, the second Xaa is Ser, and the third                         Xaa is Arg.                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                       AspXaaAspHisXaaIleXaa                                                          5                                                                              (2) INFORMATION FOR SEQ ID NO: 9:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (D) OTHER INFORMATION:                                                         Xaa is Pro or Ala.                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:                                       XaaThrAlaAspGluLysIle                                                          5                                                                              (2) INFORMATION FOR SEQ ID NO: 10:                                             (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 7 amino acids                                                      (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (D) OTHER INFORMATION:                                                         Xaa is Thr, His or Ser.                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:                                      AspGluSerHisTyrPheXaa                                                          5                                                                              __________________________________________________________________________ 

We claim:
 1. Isolated, dimeric FAPα molecule, having a molecular weight of about 170 kilodaltons as determined by SDS-PAGE, wherein said dimeric FAPα molecule is capable of degrading extracellular matrix proteins.
 2. The dimeric FAPα molecule of claim 1, wherein each monomer of said dimeric FAPα molecule consists of the amino acid sequence of SEQ ID NO:
 2. 3. The dimeric FAPα molecule of claim 1, produced recombinantly.
 4. The dimeric FAPα molecule of claim 3, produced by a eukaryotic cell. 